Background
Currently, most malaria control programmes include vector control, early diagnosis and effective treatment of clinical cases [
1]. However,
Plasmodium falciparum resistance to anti-malarial drugs is one of the main challenges for malaria control in endemic countries since resistant parasites are widespread and continue to evolve in response to the selective pressure applied [
2‐
5]. Chloroquine (CQ) and sulfadoxine–pyrimethamine (SP) had to be discontinued for clinical malaria treatment following increased morbidity and mortality associated with resistance in the past decades. The same could happen in the near future to artemisinin-based combination therapy (ACT), the current first-line therapy, if an alternative would be available [
4‐
8]. Resistance to CQ and SP arose in South-East (SE) Asia and spread to Africa [
9‐
11]. Similarly, concern about the possible expansion or emergence of resistance to artemisinin or to its partner drug in the ACT in Africa has been raised due to parasites with increased clearance time spreading throughout SE Asia [
5,
9,
10,
12,
13]. Due to lack of an alternative anti-malarial drug with the same level of efficacy and tolerability at present and in order to achieve successful disease control and eradication, it is fundamental to understand the prevalence and geographical distribution of drug resistance. This requires (1) having up-to-date information on efficacy of these therapies in different areas, (2) establishing an early intervention system and (3) understanding more about the principles of spread of resistance in different areas [
2,
3,
14].
Resistance surveillance may be done in several different ways: in vivo, ex vivo/in vitro and by mapping molecular markers [
5,
15]. In vivo studies, such as drug efficacy trials, are the gold standard [
7,
16,
17] where resistance is characterized by treatment failure or delayed parasite clearance in patients [
4,
16,
18]. They are relatively easy to standardize and do not require complex equipment [
5]. However, drug efficacy trials are difficult to carry out due to the need of patient follow up (at least 28 days or 42–63 days for drugs with longer half-lives), financial costs, ethical clearance processes and the added logistical challenges in low transmission settings, such as a small number of people in treatment [
3,
5,
17]. In ex vivo/in vitro resistance studies, parasites are extracted from human blood, grown in culture and exposed to drugs [
5,
15]. These studies provide remarkable information on the parasite’s susceptibility, which is defined by measuring growth or replication in the presence of different concentrations of these anti-malarial drugs [
5,
15,
17]. Their main advantage is that they allow to gather information on drug susceptibility to individual drugs while avoiding the confounding effects of in vivo studies, such as host immunity or pharmacokinetics [
15,
17]. However, ex vivo studies often present difficulties in the comparison of data between different laboratories due to variations among protocols and different criteria when accepting or rejecting data, as its interpretation is mainly based on visual inspection of dose–response curves [
15].
Finally, studies of molecular markers of resistance in parasites from human blood samples are the most commonly used for resistance surveillance. These methods are reliable, timely, cost-effective, quantitative and scalable [
3,
5,
16]. Moreover, they are relatively easy to implement and interpret and provide useful information on the spread of known resistance markers [
5,
17]. Although the presence of these resistance markers is linked to an increased treatment failure, extrapolation to in vivo therapeutic [
5,
16,
17,
19] and preventive [
20] efficacy is still challenging, added to a lack of methodological standardization [
17]. In addition, both ex vivo and molecular markers studies require trained personnel and specialized laboratory facilities [
5,
17], although these facilities are becoming more common in malaria endemic countries. A limiting step in all of the above mentioned surveillance strategies is that they are dependent on participation and blood sampling of human subjects. Hence the development and execution of such studies may be expensive, time consuming and need to be ethically justified. Consequently, there are typically a relatively small number of well-studied sites in endemic areas due to logistical and financial limitations [
4].
However, the parasite’s life cycle involves the successive infection of another host besides humans: female
Anopheline mosquitoes. The collection of these vectors does not require complex and invasive tools, medical training or ethical considerations and it has been shown that mosquito stage malaria parasites are useful to perform drug-resistance epidemiological studies [
21]. Hence, a more cost-effective alternative to genetic screening of parasites in human blood would be to screen these parasites inside their vector to identify and detect the prevalence of resistant mutants in malaria endemic areas by mapping molecular markers of resistance [
3,
7,
22].
Molecular genotyping techniques have been shown to be useful in epidemiological monitoring of resistant
P. falciparum present in the vectors [
22,
23]. PCR–RFLP (polymerase chain reaction–restriction fragment length polymorphism), the traditional molecular genotyping technique for monitoring drug resistance, while relatively easy, economic [
24] and fast [
7] compared to other molecular techniques, does not allow for the discovery of novel genetic polymorphisms since it targets predefined polymorphisms [
5,
25]. Furthermore, it has relatively low sensitivity and may lead to results that cannot be directly compared between studies due to different fragment sizing that can be obtained from the same molecular marker [
5,
7,
16,
24,
25]. Sanger sequencing, a newer method and the gold standard, facilitates this, but its application to large-scale surveillance is limited by low throughput; its reagents relatively higher costs, which are directly proportional to the number of specimens genotyped; and the inability to detect polymorphisms at minor frequencies especially in high transmission areas [
3,
5,
16]. Next Generation Deep Sequencing (NGS) is the latest technique. Although it requires trained staff [
17], as all other molecular techniques, it can potentially overcome most drawbacks of other molecular genotyping methods and allow detection of novel mutations and minority variant genotypes in mixed infections and quantification of allele frequencies in mixed genotypes, which are usually classified as mutant and, therefore, avoid neglecting the presence of wild-type parasites [
3,
5,
26‐
28].
Moreover, it permits higher throughput, sensitivity, resolution and scalability by pooling all samples, allowing gathering data on the frequency of resistance alleles in a certain area [
16,
17,
28]. It is, therefore, important to consider whether the most relevant resistance data is on the level of prevalence of resistance (number of individuals infected with a parasite containing resistance marker) or overall allele frequency. To understand the evolutionary dynamics, allele frequencies need to be assessed in order to determine how fast an allele is spreading through a population. Standard surveillance techniques do not allow to obtain information on frequency of resistance alleles in the parasite population. However, NGS has been identified as a method to determine resistant allele frequencies in a population [
3] and can potentially benefit the identification of circulating drug-resistant alleles of individual parasites before they are even selected by drug pressure [
17,
26].
Here, the aim was to test whether NGS for P. falciparum resistance marker detection in mosquito samples is feasible and if it is a suitable, economic and high-throughput alternative for molecular resistance surveillance.
Discussion
Here, using a Next Generation Sequencing platform, mutant allele frequencies were obtained of P. falciparum parasites isolated from mosquitoes from southern Mozambique. Similar allele frequencies of resistance markers were found with NGS compared to the prevalence of markers obtained with the gold standard Sanger sequencing. These resistance data obtained from mosquitoes involved a simpler and non-invasive sample collection, and the NGS approach allowed for high-throughput analyses leading to epidemiologically more relevant allele frequencies as opposed to resistance prevalence. Therefore, this mosquito-based NGS approach is a valuable drug resistance marker surveillance tool to fill in the large geographical gaps in resistance surveillance.
Both Sanger and NGS reflected 100% of prevalence of the wild-type allele of the
k13 propeller gene in positions Y493H, R539T, I543T and C580Y, polymorphisms associated with artemisinin resistance [
36]. This finding along with other studies supports the notion that artemisinin-driven selection on the
k13 locus is still absent in Africa [
23,
36]. However, Sanger sequencing revealed one not yet described polymorphism on the
k13 gene in one of the head/thorax samples in position S624L. Although the relevance of this single observation is uncertain, it has been shown that new point mutations frequently appear worldwide in the
k13 locus [
36,
51‐
54] and, even though not being strongly selected at this time outside SE Asia, they have the potential to enable resistance to rapidly emerge in the future [
13,
55]. Particularly with the recent observations of an independent emergence of the C580Y point mutation in Guyana [
56] and the reporting of an artemisinin-resistant
P. falciparum with a previously unreported SNP in position M579I contracted in Equatorial Guinea [
54],
k13 molecular surveillance is of critical importance. With newly arising mutations starting at low frequencies in a population, allele frequency estimates, rather than prevalence estimates, are more reliable [
36,
57]. This study further confirmed the decades long increase of wild-type
pfcrt parasites in the area [
23,
39], with no mutations at all observed on this locus compared to 85% presence of K76T mutation in 1999 [
58]. As previously observed by Gupta and colleagues [
23], Sanger sequencing analyses of
pfmdr1 revealed that more than half (53.1%) of the positive for
P. falciparum mosquitoes tested exhibited Y184F mutation, including 32% of the total that accounts for mixed (wild-type and mutant) infections. NGS analyses were very similar with 49.3% frequency of this same point mutation. Furthermore, new possible point mutations appeared during Sanger sequencing analyses of
pfmdr1 in positions T1069T, T1071V and S1075N in five different mosquito abdomen samples. Positions T1071V and S1075N have not been previously described and were polymorphic in only one sample each. However, position T1069T has been previously reported [
23] and showed the same mutation along 4 mosquito samples, which could maybe indicate a plausible novel mutation. On a different note, SP resistance linked to mutations in the
pfdhps and
pfdhfr genes in Africa is widespread [
9]. In the study area, quintuple mutations of
pfdhps and
pfdhfr were nearly fixed. Yet, positions C50R/S and I164L of
pfdhfr and S436F/A, A581G and A613T/S of
pfdhps still remain wild-type according to results of both methods. Although it has been observed that
pfdhfr polymorphism frequencies in mosquitoes may differ from those in humans [
59], overall, our observations are within a similar range to those numbers obtained from human blood samples in other studies [
60].
Sanger sequencing and NGS approaches gave overall very similar resistance markers estimates, in spite of some minor discrepancies. As previously mentioned, some of these discrepancies are due to the fact that Sanger will provide with prevalence approximations while NGS will measure allele frequency. While prevalence of resistance on a human subject level may inform treatment choice, on an epidemiological level the parameter of interest is allele frequency. Furthermore, frequency estimates also allow to capture minority emerging genotypes in mixed infections, which would be missed with prevalence analyses that do not detect mixed alleles below a threshold of 10–20% [
3,
60,
61]. On the flipside, however, prevalence analysis would be more sensitive for the detection of mutants that are at a higher frequency within a given sample but at low frequency on a population level. The latter, however, would be less likely to occur for novel emerging mutations. The NGS approach allows for pooling of samples too, which reduces cost and performance time without compromise in information since the objective is population-level allele frequency [
3]. Moreover, pooled deep sequencing offers high read coverage and sequencing depth and permits to increase sample size, as previously seen in other studies [
3,
26,
27]. It also provides the means to monitor whole genes, which is necessary for those with multiple point mutations associated with resistance and which would allow the detection of novel SNPs usually not analysed with other detection techniques. Moreover, results from this study show that pooled deep sequencing of infected mosquito samples is a more suitable alternative to pooled human blood samples deep sequencing. It does not require specialized personnel to draw blood from patients and it avoids complex ethical requirements and visiting expeditions, which can sometimes be very challenging in low-income settings. Since data obtained from mosquitoes has been shown to correlate well with data from humans [
23,
36,
39,
60], mosquitoes could be used as a sentinel group for resistance surveillance purposes.
A relatively high
P. falciparum infection rate (23.6%) was observed in these mosquitoes collected in southern Mozambique, an area with high level of malaria transmission (between 100 and 200 cases per 1000 population) [
29]. Of note however, was that extracts from head/thorax (6%) and abdomen (18.3%) were for unclear reasons significantly lower than isolates from whole mosquitoes. Further studies are also needed to confirm if only abdomens should be screened as according to our results they presented a higher positivity rate. However, it should be taken into account that there is the possibility to a change in allele proportions between head/thorax and abdomen portions. Nevertheless, as mentioned beforehand, for allele frequency purposes, however, a pooling strategy of whole mosquitoes would be adequate, such as previously demonstrated for the detection of dengue [
62] and Ross River viruses [
63], though with the limitation that any pooling strategy could bias results towards higher density infections. Interestingly, more
P. falciparum positive mosquitoes appeared to be captured by miniature light traps than early morning collections using manual aspiration (Table
1). An intriguing hypothesis is that this could be due to behavioural manipulation: it is thought that
Plasmodium-infected humans present an increased attractiveness to the arthropod vector [
64] and that those mosquitoes infected with
P. falciparum are more attracted to humans [
65]. Moreover, it has explicitly been shown that
Aedes aegypti infectious with
Plasmodium gallinaceum present an increased host-seeking behaviour [
66]. Because miniature light traps are located in close proximity to people sleeping under LLINs, this could explain the higher frequency of
P. falciparum positive mosquitoes captured by the miniature light traps.
Although the proposed surveillance tool of NGS of
P. falciparum isolates from mosquitoes is promising, there are some caveats. First, the validity of extrapolation of resistance marker frequency in mosquitoes to human population needs to be confirmed. Detection of resistance markers could be more sensitive in the human host—when de novo mutants could have been selected—instead of during the mosquito life cycle, when negative selection against mutants could occur. However, evidence for this effect is sparse (reviewed in [
67]). Of note is that frequency of resistance surveillance in the mosquito vector is arguably a more relevant measure of resistance epidemiology as anti-malarial resistance is more likely to be transmitted than acquired (reviewed in [
68]). Second, this study is based on a single mosquito species,
Anopheles funestus, an indoor-biting highly antropophylic vector, and the extent to which different vector species carry different
P. falciparum genotypes, and hence bias the allele frequency, is unknown. However, it has been observed that different
Plasmodium genotypes are randomly distributed [
69]. Third, these mosquitoes were collected in a relatively high transmission area. The approach may be less cost-effective in a low transmission area when a larger number of mosquitoes need to be screened and NGS will be less capable of detecting low-density infections. However, this is a general issue for resistance surveillance in low transmission areas, irrespective of using human or mosquito samples. Pooled screening approaches would significantly reduce this cost to more efficiently identify
P. falciparum infected specimens with the caveat that pooling could bias results to high density mosquito samples. Fourth, as with many molecular approaches, this Illumina sequencing approach only allows the detection of known SNPs. Other approaches will be needed to detect novel mutations and gene duplications. Finally, NGS is a relatively new technique linked to uncertainty due to errors in alignment, base calling or filtering [
50]. For example, unexpected mutations in positions S436F/A, A437G, A581G and A613T/S of
pfdhps in the 3D7 control were found (see Additional file
1: Table S6) and statistically significant differences among Sanger and NGS results in
pfmdr1 position N86Y/F,
pfdhps position A437G and
pfdhfr position C50R/S. These statistically significant differences could possibly be overcome by moving to an individual NGS approach rather than a pooled one. However, they could still be either a false negative or positive by Sanger sequencing or by NGS or a distinction between prevalence and frequency. It is, therefore, imperative to account for uncertainty linked to NGS and to try to reduce it. There are different strategies to do so, although one must accept that there is not a perfect combination and that the fast development of bioinformatic tools means that recommendations may change very rapidly but also improve [
50].
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